Novel approaches for prediction and early diagnosis of lung cancer are based on mutation analysis and the detection of atypical changes in the cell structure (Lacroix et al., Expert Review Mol Diagn (2008) 8(2) 167-178). The main mutations which are associated with lung cancer are Epidermal Growth Factor Receptor (EGFR) mutations and V-Ki-ras2 Kirsten ras sarcoma viral oncogene homolog (KRAS) mutations. The EGFR is a 170-kD transmembrane protein which is a member of the receptor kinase family. EGFR is widely expressed in several malignancies including lung, breast, colon, esophageal and others. Activating mutations in exons 18-21 of EGFR were initially identified in non-small cell lung carcinoma (NSCLC) patients with clinical response to gefitinib (Lynch et al., N Engl J Med (2004) 350(21) 2129-2139; Paez et al., Science (2004) 304(5676) 1497-1500). Patients with EGFR mutations have a greater response rate to EGFR-targeted therapy than patients with wild type EGFR or unknown mutation status (10-20%) (Riely et al., Clin Cancer Res (2006) 12(3 Pt 1) 839-844). Recent studies suggest that EGFR-targeted therapy is preferred over chemotherapy in chemo-naïve patients with EGFR mutations. Mok et al. (N Engl J Med 2009 361) showed that the presence of an EGFR mutation was found to be a robust predictor of improved progression-free survival with gefitinib, as compared with carboplatin-paclitaxel. Peled et al. (Ther Adv Med Oncol (2009) 1(3) 137-144) discloses that there are several biomarkers including EGFR mutation status, EGFR protein expression, EGFR gene copy number, and a serum proteomin marker that are able to direct and predict the result of EGFR-related therapies in NSCLC.
Additional mutations that have been suggested for the direction of anti-cancer therapy in lung cancer are KRAS mutations. KRAS is an important downstream mediator of EGFR signaling and harbors an activating mutation in codon 12 or 13 (exon 2) in approximately 10-30% of NSCLC cases. EGFR and KRAS activating mutations are almost always mutually exclusive. In NSCLC, KRAS mutation has been demonstrated to be associated with poor prognosis and is thus a negative prognostic factor, which needs to be taken into account when the predictive performance/response is assessed.
The echinoderm microtubule-associated protein like-4/anaplastic lymphoma kinase (EML4-ALK) translocation results from a small inversion within chromosome 2p and has been associated with approximately 5% to 13% of lung cancers (Chiarle et al., Nat Rev Cancer (2008) 8 11-23; Mano, Cancer Sci (2008) 99 2349-2355; Shaw et al., J Clin Oncol (2009) 27 4247-4253; and Wong et al., Cancer (2009) 115 1723-1733). The resulting EML4/ALK fusion protein possesses potent oncogenic activity through the Ras/Raf, PI3K/Akt, and JAK/STAT pathways, which are also stimulated by EGFR (Soda et al., PNAS USA (2008) 105 19893-19897; and Solomon et al., J Thorac Oncol (2009) 4 1450-1454). In transgenic mice expressing the EML4-ALK fusion protein in lung alveolar epithelial cells, hundreds of tumors develop within a few weeks of birth. The tumors can be effectively inhibited by small molecules that target ALK, supporting a role for EML4-ALK as a promoter of lung tumorigenesis (Soda et al., PNAS USA (2008) 105 19893-19897). Epidemiologically, patients with tumors expressing the EML4-ALK fusion tend to be younger males, have a never/light smoking history and have tumors with adenocarcinoma histology, specifically the signet ring subtype (Shaw et al., J Clin Oncol (2009) 27 4247-4253). Patients whose tumors express the EML4-ALK fusion often do not respond to EGFR tyrosine kinase inhibitors (Shaw et al., J Clin Oncol (2009) 27 4247-4253). Activity of an EML4-ALK inhibitor was observed in a phase I trial in patients with NSCLC whose tumors were EML4-ALK-positive. Other agents that target the ALK pathway are currently in development (Solomon et al., J Thorac Oncol (2009) 4 1450-1454).
In order to determine the existence of a mutation/genetic abnormality in a cancer cell, DNA gene sequencing along with Polymerase Chain Reaction (PCR) amplification is required. Other methods include gene array analysis that is based on RNA sequencing, and immunohistochemistry techniques in which antibodies to a specific protein are used. However, such procedures are expensive, time consuming, require specialists in analyzing the results and are not suitable for non-resectable tumors. Another evolving technique is circulating DNA which can be detected in the plasma and serum of patients (Gautschi et al., J Clin Oncol (2005) 23(36) 9105). The levels of circulating DNA are associated with a poor Tumor-specific DNA alterations (such as loss of heterozygosity), promoter methylation, and KRAS and EGFR mutations. New techniques for capturing circulating tumor cells enable the detection of EGFR-activating mutations, and the drug-resistance allele T790M. Such techniques appear to be more sensitive than those for capturing circulating DNA. Furthermore, a decline in the number of circulating tumor cells was associated with tumor response to radiography (Maheswaran et al., Engl J Med (2008) 359 (4) 366). However, all available conventional methods for the detection and identification of cancer genetic mutations lack the requisite sensitivity to enable clinical utility (Mack et al., J. Thorac. Oncol. (2009) 4(2) 1466). In addition, they are significantly affected by the presence of stromal (and/or connective) tissue in the specimen taken. More importantly, cancer cells change their characteristics over time; new mutations occur frequently in the metastasic lesions and/or in the primary area over time. These mutations require frequent monitoring using invasive procedures.
Dysplasia or dysplastic changes are atypical changes in the nuclei of cells, the cytoplasm, or the growth pattern of cells. These changes which vary from subtle changes to pronounced changes are considered pre-cancerous conditions. Dysplasia is characterized by four major pathological microscopic changes as follows: anisocytosis (cells of unequal size), poikilocytosis (abnormally shaped cells), hyperchromatism (degeneration of cell nuclei, which become filled with particles of pigment (chromatin)), and the presence of mitotic figures (an unusual number of cells which are currently dividing). As the risk for cancer increases with the progression of the dysplasia, detecting dysplasia allows focusing on the high-risk cohort and defining the group for specific follow up and/or treatment, e.g. routine bronchoscopies, chemoprevention therapy, etc.
Atypical alveolar hyperplasia (AAH) has recently been described in human lungs in association with primary lung cancer, particularly adenocarcinoma. Unlike proximal bronchogenic carcinoma, peripheral (parenchymal) adenocarcinoma of the lung does not have a well-recognized progenitor lesion. Epidemiological morphometric, and cytofluorometric data in the literature suggest that AAH is a candidate pre-malignant entity.
Several investigative tools have been proposed for the detection of pre-invasive lesions and early lung cancers. Spiral CT scanning is not suitable for detecting such findings in the central airways, especially the early stages of pre-invasive squamous cell carcinoma, which account for 17-29% of all lung cancers (Jemal et al., CA Cancer J Clin (2009) 59(4), 225-249). White light bronchoscopy (WBL) is also considered insufficient for detecting such lesions. Auto fluorescence bronchoscopy (AFB) uses a helium-cadmium laser to illuminate the bronchial mucosa with 442-nm light. The red and green autofluorescence emitted light is captured by photoamplifier camera and is presented as green for normal areas and red brown for abnormal areas. During the last few years, a new bronchoscope, the Narrow-band Imaging Bronchoscope (NBI), has been evaluated to detect bronchial dysplasia and carcinoma in situ. The NBI uses two bandwidths of light: 390-445 nm (blue) light that is absorbed by superficial capillaries and 530-550 nm (green) light that is absorbed by blood vessels below the mucosal capillaries. These narrow bandwidths reduce the scattering of light and enable enhanced visualization of blood vessels (Herth et al., J Thorac Oncol (2009) 4(9) 1060-1065) thus increasing the sensitivity in detecting bronchial dysplasia and metaplasia. EP 1447043 discloses an apparatus for imaging diagnosis of tissue using diagnostic white light endoscopy (DWLE) and diagnostic auto fluorescence endoscopy (DAFE).
Several predictive and prognostic markers have been suggested for the identification of dysplastic conditions and early diagnosis of lung cancer (Coate et al., Lancet Oncol (2009) 10 1001-1010). Specific genetic abnormalities that increase the risk for cancer as well as those which occur in cancerous tissue (and not in normal tissue) were found to be significant for the therapy through specific pathway that might be associated with the carcinogenesis of the tumor. For example, Soda et al. (Nature (2007) 448 561-567) discloses that a small inversion within chromosome 2p results in the formation of a fusion gene comprising portions of the echinoderm microtubule-associated protein-like 4 (EML4) gene and the anaplastic lymphoma kinase (ALK) gene in NSCLC cells. WO 2009/118205 discloses means for the diagnosis, prognosis and/or treatment monitoring of lung cancer or bronchial dysplasia, and the use thereof for predicting and monitoring therapeutic intervention in dysplasia or cancer patients using at least one peptide. WO 2001/042504 discloses the detection of specific extracellular nucleic acid derived from mutant oncogenes or other tumor-associated DNA in plasma or serum fractions of human or animal blood associated with neoplastic, pre-malignant or proliferative disease. EP 1416278 discloses a method for improved diagnosis of dysplasias based on simultaneous detection of INK4a gene products and at least one marker for cell proliferation.
Volatile Organic Compounds
Volatile organic compounds (VOCs) are small organic molecules released during cellular metabolic processes. Patterns of VOCs are known to be used as biomarkers of various diseases. In exhaled breath of patients with cancer, elevated levels of certain VOCs including volatile C4-C20 alkane compounds, specific monomethylated alkanes as well as benzene derivatives were found.
In recent years many attempts have been made to identify one specific pattern of volatile organic compounds (VOCs) in the breath of lung cancer patients. Phillips et al. (Lancet (1999) 353 1930-1933) used discriminant analysis to detect a combination of 22 breath VOCs as the “fingerprint” of lung cancer. Phillips et al. (Chest (2003) 123 2115-2123) then used a predictive model employing 9 VOCs which was found to exhibit sufficient sensitivity and specificity to be used as screen for lung cancer. In a more recent study, Phillips et al. (Cancer Biomarkers (2007) 3 95-109) described the use of multi-linear regression and fuzzy logic to analyze breath samples of lung cancer patients. This study provided a set of 16 VOCs as the major identifiers of primary lung cancer in breath. The use of weighted digital analysis to select 30 breath VOCs as candidate biomarkers of primary lung cancer was then employed (Phillips et al., Clinica Chimica Acta (2008) 393 76-84).
Yu et al. (Sensors, Proceedings of IEEE (2003) 2 1333-1337) used an electronic nose device with capillary column GC and a pair of surface acoustic wave sensors to detect 9 VOCs as markers for lung cancer. Chen et al. (Meas Sci Technol (2005) 16 1535-1546) used a set of 11 VOCs to calibrate sensors array based on surface acoustic wave to diagnose lung cancer patients. In another study, Chen et al. (Cancer (2007) 110 835-844) identified 4 special VOCs that were found to exist in all culture mediums of lung cancer cells and can be used as markers of lung cancer. Di Natale et al. (Biosensors and Bioelectronics (2003) 18 1209-1218) used an array of non-selective gas sensors for detecting various alkanes and benzene derivatives as possible candidate markers of lung cancer. Gordon et al. (Clin Chem (1985) 31(8) 1278-1282) used breath collection technique and computer-assisted gas chromatography/mass spectrometry to identify several volatile organic compounds in the exhaled breath of lung cancer patients which appear to be associated with the disease. Song et al. (Lung Cancer (2009) 67 227-231) reported that 1-butanol and 3-hydroxy-2-butanone were found at significantly higher concentrations in the breath of the lung cancer patients compared to the controls. These two VOCs are thus potential biomarkers useful for diagnosing lung cancer. O'neill et al. (Clinical Chemistry (1988) 34(8) 1613-1617) reported a list of 28 VOCs found in over 90% occurrence in expired-air samples from lung cancer patients. Wehinger et al. (Inter J Mass Spectrometry (2007) 265 49-59) used proton transfer reaction mass-spectrometric analysis to detect lung cancer in human breath. Two VOCs were found to best discriminate between exhaled breath of primary lung cancer cases and control. Gaspar et al. (J Chromatography A (2009) 1216 2749-2756) used linear and branched C14-C24 hydrocarbons from exhaled air of lung cancer patients, smokers and non-smokers for multivariable analysis to identify biomarkers in lung disorders. Poli et al. (Respiratory Research (2005) 6 71-81) showed that the combination of 13 VOCs allowed the correct classification of cases into groups of smokers, patients with chronic obstructive pulmonary disease, patients with non-small cells lung cancer and controls. Recently, Poli et al. (Acta Biomed (2008) 79(1) 64-72) measured VOC levels in exhaled breath of operated lung cancer patients, one months and three years after surgical removal of the tumor. Peng et al. (Nature Nanotech (2009) 4 669-673) identified 42 VOCs that represent lung cancer biomarkers using gas chromatography/mass spectrometry.
In addition to the many studies that were aimed at identifying VOCs indicative of lung cancer from breath samples, Filipiak et al. (Cancer Cell International (2008) 8 17) disclosed a list of 60 substances observed in the headspace of medium as well as in the headspace of lung cancer cell line CALU-1. Barash et al. (Small (2009) 5(22) 2618-2624) discloses a list of 15 VOCs which were found in the headspace of non-small cell lung carcinoma samples. These VOCs were not found in the headspace of control cell lines. Sponring et al. (Anticancer Res (2009) 29(1) 419) found that at least two substances, 2-methylpentane and 2-ethyl-1-hexanol, can be released from the NCI-H2087 lung cancer cell line. These studies cumulatively provided over 150 VOCs as potential lung cancer biomarkers in breath samples.
WO 2000/041623 discloses a process for determining the presence or absence of a disease, particularly breast or lung cancer, in a mammal, comprising collecting a representative sample of alveolar breath and a representative sample of ambient air, analyzing the samples of breath and air to determine content of n-alkanes having 2 to 20 carbon atoms, inclusive, calculating the alveolar gradients of the n-alkanes in the breath sample in order to determine the alkane profile, and comparing the alkane profile to baseline alkane profiles calculated for mammals known to be free of the disease to be determined, wherein finding of differences in the alkane profile from the baseline alkane profile being indicative of the presence of the disease.
WO 2010/079491 to one of the inventors of the present invention discloses a set of volatile organic compounds indicative of lung cancer, and methods of diagnosing or monitoring lung cancer progression using such set of volatile organic compounds.
There is an unmet need for the identification of genetic abnormalities such as EGFR and KRAS mutations and/or ALK-ELM translocation and for the identification of atypical changes in lung/bronchial cells for the prediction, early diagnosis and targeted treatment of lung cancer.